GSM7089445: CTCF-AID_ZNF143KO_CTCF-Degron; Homo sapiens; Hi-C
About 5 million cross-linked cells were lysed with lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% NP-40) to obtain nuclei. Nuclei were incubated with SDS and quenched by adding Triton X-100. Samples DNA was digested with MboI (NEB R0147M) for overnight at 37℃. After heat inactivation at 62℃, restriction fragment overhangs were filled and marked with biotin-14-dATP (Thermo 19524016), dCTP, dTTP, dGTP, and DNA Polymerase I, Large (Klenow) Fragment (NEB M0210L). After Proximity ligation using T4 DNA ligase (NEB M0202S), samples were reverse cross-links at 68℃ for overnight. DNA samples were precipitated with ethanol and then sonicated to fragmentize DNA. DNA fragment size selection was performed by using AMPure XP Beads (Beckman A63881), and 300-500 bp DNA fragments were used for the next step. M-280 Streptavidin beads (Thermo 11206D) were used to select DNA fragments marked with biotin. Library construction steps were performed while DNA samples were still bound to the bead interval with strict washing. NEB end-repair module (NEB E6050S) was used to blunt DNA end and dA-Tailing DNA reaction performed with Klenow exo- (NEB M0212S). Illumina U-type adaptors were adopted to add adaptor for DNA, and USER enzyme (NEB M5505S) was used for adaptor cleavage. After 10-12 of cycles PCR amplification, DNA samples were purified with AMPure XP Beads (Beckman A63881). In situ Hi-C